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To clarify the role of the C5a/C5aR (C5a receptor) and C5b-9 pathways in macrovascular thrombosis (MAT) and renal microthrombosis (MIT), 73 renal biopsy-proven complement-mediated thrombotic microangiopathy (C-TMA) patients were enrolled; 9 patients with pure MAT and 13 patients with pure MIT were selected for further study. Twenty-five external C-TMA patients were selected as the validation cohort. Plasma C5a and sC5b-9 (soluble C5b-9) levels were significantly higher in patients with MAT than in those with MIT (P = 0.008, P = 0.041, respectively). The mean optical density of C5aR1 in the kidney was significantly higher in MAT patients than in those with MIT (P < 0.001). Both urinary sC5b-9 levels (MIT: P < 0.001, MAT: P = 0.004) and renal deposition of C5b-9 (MIT: P < 0.001, MAT: P = 0.001) were significantly higher in C-TMA patients compared to normal control, but were similar between MAT and MIT groups. In the correlation analysis within 22C-TMA patients, urinary sC5b-9 levels and renal deposition of C5b-9 were positively correlated to renal MIT formation (P = 0.009 and P = 0.031, respectively). Furthermore, the renal citrullinated histone H3 (CitH3)- and neutrophil elastase (NE)-positive area ratios were both significantly higher in the MAT group than in the MIT group (P = 0.006 and P = 0.020, respectively). Therefore, the local C5b-9 and C5a/C5aR1 pathways might have differential contributions to MIT and MAT formation in the disease., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11., (© 2023. The Author(s).)

Background: Surface modifications of dental implants have gained attention during several years and the thrombotic response from blood components with these materials has become more important during recent years., Purpose: The aims of this study were to evaluate the thrombogenic response of whole blood, in contact with clinically used dental surfaces, Sandblasted Large grit Acid etched titanium (SLA) and Sandblasted Large grit Acid etched, and chemically modified titanium with hydrophilic properties (SLActive)., Methods: An in vitro slide chamber model, furnished with heparin, was used in which whole blood came in contact with slides of the test surfaces. After incubation (60-minute rotation at 22 rpm in a 37°C water bath), blood was mixed with ethylenediaminetetraacetic acid (EDTA) or citrate, further centrifuged at +4°C. Finally, plasma was collected pending analysis., Results: Whole blood in contact with surfaces resulted in significantly higher binding of platelets to the hydrophilic surface, accompanied by a significant increase of contact activation of the coagulation cascade. In addition, the platelet activation showed a similar pattern with a significant elevated release of β-TG from platelet granule., Conclusions: The conclusion that can be drawn from the results in our study is that the hydrophilic modification seems to augment the thrombogenic properties of titanium with implications for healing into bone of, that is titanium dental implants., (© 2011 Wiley Periodicals, Inc.)

Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the approximately 150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of approximately 75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a K(d) of approximately 3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity.

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation.

Background: An injection of anti-Fx1A antibodies in rats leads to passive Heymann nephritis (PHN), a model of membranous nephropathy. Fx1A is a crude extract of renal cortex that contains megalin as a principal component. However, when rats are given anti-megalin antibodies, abnormal proteinuria does not occur. Because of the established complement dependence of PHN, we hypothesized that antibodies neutralizing complement regulatory proteins in the rat glomerulus also were required to induce PHN. Two likely targets are Crry and CD59, proteins abundant on the rat podocyte and contained within Fx1A that inhibit the C3 convertase and C5b-9 assembly, respectively., Methods: Rats were injected with anti-megalin monoclonal antibodies, followed by anti-Crry and/or anti-CD59 F(ab')(2) antibodies five days later. In a second group of experiments, rats were injected with anti-Fx1A or anti-Fx1A immunodepleted of reactivity against Crry and/or CD59., Results: In the setting of podocyte-associated anti-megalin monoclonal antibodies, simultaneous neutralization of Crry and CD59 function led to the development of significant proteinuria (11.0 +/- 2.1 mg/day, P < 0.001 vs. all other groups). In contrast, animals that had neither or only one of these complement regulators inhibited had normal urinary protein excretion (< or =6 mg/day). In animals given anti-Fx1A depleted of anti-Crry and/or anti-CD59, all groups developed typical PHN, characterized by heavy proteinuria and extensive glomerular deposition of C3 and C5b-9., Conclusion: Crry and CD59 play an important role in restraining complement-mediated injury following subepithelial immune complex deposition; however, in PHN, their regulatory capacity is overwhelmed.

Background: Complement activation has recently been implicated as a contributing factor to early and late allograft dysfunction in cardiac transplantation. The current study was designed to determine whether measurement of plasma complement fragments C4d and SC5b-9 would be useful in detecting acute rejection or accelerated graft atherosclerosis (AGA) in cardiac allograft recipients., Methods: We measured complement activation products, C4d (classical pathway) and SC5b-9 (terminal pathway), at the time of routine endomyocardial biopsy in heart transplant recipients. Ten patients in the immediate posttransplantation period (0-100 days) and 19 patients more than 6 months after transplantation were studied., Results: No correlation was found between plasma levels of complement activation fragments and the presence of biopsy-proven acute allograft rejection or AGA (assessed by coronary angiography). However, plasma C4d and SC5b-9 were significantly elevated in 9 of 10 and 7 of 10 patients, respectively, in the immediate posttransplantation period. This was followed by progressive decrease in the levels of C4d and SC5b-9 fragments during the first 4-6 weeks after transplantation., Conclusion: We conclude that measuring plasma levels of fragments C4d and SC5b-9 is not a useful noninvasive method for detecting acute rejection or AGA after heart transplantation. However, this study provides further evidence that early complement activation after heart transplantation may play a pathogenic role in allograft injury.

Background: Deranged Na(+) homeostasis in skeletal muscle is closely associated with excessive complement activation that is encountered during sepsis. Recent evidence suggests that soluble C5b-9 complexes (SC5b-9), which are readily detected in plasma during sepsis and have long been considered irrelevant nonmembrane binding end products of complement activation, may have numerous biologic effects. The purpose of this study, therefore, was to determine the effects of SC5b-9 on myocellular ion homeostasis and its mechanism(s) of action., Methods: Hindlimb fast-twitch extensor digitorum longus (EDL) was freshly isolated from rats weighing 50 to 70 g and then incubated at 30 degrees C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37 degrees C for 60 minutes) as a source of SC5b-9. Zymosan particles were removed by centrifugation after activation to exclude any noncomplement direct effects. Heat-inactivated rat serum (56 degrees C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 microg/mL), in Ca(2+)-free KHB, with thapsigargin (0.3 or 3 micromol/L), or with ouabain (0.01, 0.1 or 1 micromol/L) before and/or during incubation with 10% zymosan-activated or heat-inactivated rat serum. Intracellular Na(+) and K(+) contents ([Na(+)](i) or [K(+)](i)) of EDL muscle were determined by using flame photometry after washing in ice-cold Na(+)-free Tris-sucrose buffer. SC5b-9 in zymosan-activated human serum was determined by SC5b-9 enzyme-linked immunoassay., Results: SC5b-9 in zymosan-activated human serum significantly increased by 400% as compared with nonactivated, normal human serum. Zymosan-activated rat serum markedly increased [Na+]i without affecting [K(+)](i) in fast-twitch EDL muscle, which was completely inhibited by pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) with thapsigargin. The addition of ouabain (at micromolar concentrations) increased myocellular [Na(+)](i) and decreased myocellular [K(+)](i) in both the zymosan-activated and the heat-inactivated rat serum groups. The effects of ouabain on myocellular [Na(+)](i) and [K(+)](i) were equivalent in these 2 groups. Zymosan-activated and heat-inactivated rat serum had similar effects on myocellular [K(+)](i) in the presence or absence of pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+)., Conclusions: Zymosan-activated rat serum (presumed SC5b-9 enriched) selectively alters Na(+) homeostasis in isolated fast-twitch skeletal muscle. The mechanisms for such effects may be linked to G-proteins, Ca(2+) flux and Na(+),K(+)-adenosine triphosphatase pump binding site blockade.

The activation of complement system is an important factor participating in inflammatory, neurodegenerative, and cerebrovascular diseases. Astrocytes and neurons are able to synthesize complement components. Myelin and oligodendrocyte (OLG) activate the classical pathway of complement in vitro in the absence of antibodies. Sublytic C5b-9 in the absence of cell death induces proto-oncogenes, activates cell cycle, and enhances cell survival in OLG. In addition, C5b-9 reverses the differentiation phenotype in OLG and enhances cell survival. beta amyloid protein is an activator of the complement system and neurons are susceptible to bystander complement mediated damage. These findings indicate that complement activation and membrane assembly of C5b-9 play an important role in pathogenesis of central nervous system (CNS) disorders.

Mannan-binding lectin (MBL) is an acute phase protein which activates the classical complement pathway at the level of C4 and C2 via two novel serine proteases homologous to C1r and C1s. We recently reported that haemolysis via this lectin pathway requires alternative pathway amplification. The present experiments sought to establish the basis for this requirement, and hence focused on the activity and regulation of the C3 convertases. Complement activation was normalized between the lectin and classical pathways such that identical amounts of bound C4 and of haemolytically active C4,2 sites were present on the indicator cells. Under these conditions, there was markedly less haemolysis, associated with markedly less C3 and C5 deposited, via the lectin pathway than via the classical pathway, particularly when alternative pathway recruitment was blocked by depletion of factor D. Lectin pathway activation was associated with enhanced binding in the presence of MBL of complement control proteins C4bp and factor H to C4b and C3b, respectively, with decreased stability of the C3-converting enzyme C4b,2a attributable to C4bp. Immunodepletion of C4bp and/or factor H increased lectin pathway haemolysis and allowed lysis to occur in absence of the alternative pathway. Thus, the lectin pathway of humans is particularly susceptible to the regulatory effects of C4bp and factor H, due at least in part to MBL enhancement of C4bp binding to C4b and factor H binding to C3b.

The plasma complement system comprises several activation pathways that share a common terminal route involving the assembly of the terminal complement complex (TCC), formed by C5b-C9. The order of emergence of the homologous components of TCC (C6, C7, C8alpha, C8beta, and C9) has been determined by phylogenetic analyses of their amino acid sequences. Using all the sequence data available for C6-C9 proteins, as well as for perforins, the results suggested that these TCC components originated from a single ancestral gene and that C6 and C7 were the earliest to emerge. Our evidence supports the notion that the ancestral gene had a complex modular composition. A series of gene duplications in combination with a tendency to lose modules resulted in successive complement proteins with decreasing modular complexity. C9 and perforin apparently are the result of different selective conditions to acquire pore-forming function. Thus C9 and perforin are examples of evolutionary parallelism.

C3b and C5b deposition following complement activation, and its regulation by CD46 were studied using xenogenic Chinese hamster ovary (CHO) cells as targets and cytofluorometry. Following activation of the alternative pathway, an initial low level of C3b deposition was observed on CHO cell surfaces after a lag time of approximately 4 min. This was followed by a secondary high level of C3b deposition with a slower rate. C3b deposition was maximal within 15 min. When CD46 was expressed (B2 isoform), the kinetics of C3b deposition were essentially unchanged, but the onset of the secondary high C3b deposition was fully prevented. C5b deposition was also observed on CHO but not on CHO.CD46 cells following activation of the alternative pathway. Activation of the classical pathway on CHO and CHO.CD46 cells, using factor B-depleted human serum and anti-CHO antibodies, resulted in almost identical single-peak C3b deposition profiles. Accordingly, no regulation of C5b deposition by CD46 was evident following activation of the classical pathway. These data indicate that CD46 prevents the C3b deposition amplification loop mediated by the alternative C3 convertase and, consequently, inhibits the formation of the alternative C5 convertase. But CD46 prevents neither the spontaneous tick-over C3b deposition leading to the formation of the alternative C3 convertase nor the formation of the functional classical C3 and C5 convertases.

Large numbers of neuritic plaques (NP), largely composed of a fibrillar insoluble form of the beta-amyloid peptide (Abeta), are found in the hippocampus and neocortex of Alzheimer's disease (AD) patients in association with damaged neuronal processes, increased numbers of activated astrocytes and microglia, and several proteins including the components of the proinflammatory complement system. These studies address the hypothesis that the activated complement system mediates the cellular changes that surround fibrillar Abeta deposits in NP. We report that Abeta peptides directly and independently activate the alternative complement pathway as well as the classical complement pathway; trigger the formation of covalent, ester-linked complexes of Abeta with activation products of the third complement component (C3); generate the cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory C5b-9 membrane attack complex, in functionally active form able to insert into and permeabilize the membrane of neuronal precursor cells. These findings provide inflammation-based mechanisms to account for the presence of complement components in NP in association with damaged neurons and increased numbers of activated glial cells, and they have potential implications for the therapy of AD.

The complement resistance of Aeromonas salmonicida strains grown under conditions promoting capsule formation was investigated using well characterized strains and their isogenic mutants. Complement resistance was previously studied using the same strains growing under non-capsulating conditions. The serum resistant strains were found to activate complement, but rapidly degrade C3b preventing productive formation of the lytic complex C5b-9. Isogenic lipopolysaccharide rough mutants grown under non-capsulating conditions were serum sensitive, binding a large amount of C3b and leading to productive formation of C5b-9. When grown under conditions promoting capsule formation, these mutants were partially resistant to complement because less C3b is bound to them and also partially degraded, with a concomitant reduction in lytic C5b-9.

The occurrence of IgG, IgM, IgA, C3 and C5b-9 complement complexes on erythrocytes from 43 patients with beta-thalassaemia HbE disease was investigated. Indirect immunoradiometric assays using radioiodinated protein A were employed to quantify the individual components. We confirmed that circulating erythrocytes from thalassaemic patients contained elevated amounts of IgG, and small but significant amounts of C3. In addition, small but significant amounts of C5b-9 were detected. Levels of cell-bound IgG, C3 and C5b-9 were higher in splenectomized versus non-splenectomized patients. The presence of C5b-9 on circulating cells from five splenectomized patients was confirmed by an ELISA employing a monoclonal antibody specific for a C5b-9 neoantigen. When C5b-9 positive cells from two patients were solubilized with detergent and subjected to sucrose density gradient centrifugation, the terminal complexes sedimented as 25-40S macromolecules, thus behaving as membrane C5b-9 complexes. The presence of C8 and C9 in these high molecular weight fractions was directly demonstrated by Western blotting. These results constitute the first demonstration that circulating diseased erythrocytes may carry low numbers of potentially cytolytic C5b-9 complement complexes which may be partly responsible for the known ionic disturbances found in thalassaemic cells. Both bound C3 and C5b-9 could promote removal of diseased cells in the reticuloendothelial system.

The relationship between the presence of antiphospholipid antibodies (APA) and the production of the terminal membrane attack complex (MAC) of complement (C5b-9) was studied. Serum samples from known high positive APA patients induced platelet activation and destruction which was inhibited by heat-inactivation of the sera. The response was restored if the heat-inactivated APA-positive sera were supplemented with normal sera. Adsorption of the APA-positive sera with phospholipid (PL)-coated polystyrene beads inhibited platelet destruction. Addition of monoclonal antibody (mAb) to C5b-9 (aE11) also inhibited platelet destruction, suggesting that the APA-dependent platelet destruction might be complement-mediated. Purified APA, in the presence of normal serum, induced C5b-9 formation and binding to PL-coated beads in a dose-dependent manner as detected by flow cytometry. Prospective analysis of 200 serum samples for C5b-9 production showed that all sera testing negative for the presence of APA also tested negative for C5b-9 production. All sera with high levels of IgG binding to PL (GPL) showed evidence of C5b-9 production. Sera with low or moderate GPL values showed varying levels of C5b-9 production. These data suggest that complement may play a key role in APA-dependent platelet activation, in vivo.

Platelet-activating factor is a recognized mediator of anaphylaxis and bioincompatibility. Here, the mechanisms and the kinetics of the production of platelet-activating factor were studied in vivo during high-flux hemodialysis and in vitro in a recirculation model with polyacrylonitrile membranes, the AN-69 and the more recent SPAN, where the Na-metallilsulfonate group is partially substituted with the less polar methacrylate group. In in vivo studies, eleven patients were studied in cross over. Patients were randomly allocated to the AN-69 (5 patients) and to the SPAN membrane (6 patients) for two weeks. Measurements were made in the second week of use. After completion of the second week, the patients were switched to the other membrane for a further two weeks. Samples for leukocyte and platelet counts, PAF in whole blood or bound to platelets, the C3a des Arg and the C5b-C9 membrane attack complex as well as samples for clearances of urea, creatinine and phosphates were taken at different time intervals during treatment. PAF was detected by biological assay after methanol extraction of whole blood or of platelet pellets obtained by sequential centrifugation. C3a des Arg and the C5b-C9 fraction were detected by commercially available immunoassays. Results were analyzed by Minitab statistical package. PAF was detectable only during treatment with AN-69 but not with SPAN 1 min after start of the extracorporeal circulation in both whole blood (4.5 +/- 2.7 ng/ml) and on platelet surface (4.1 +/- 1.2 ng/ml). No statistical significant differences were observed between AN-69 and SPAN with regard to leukocyte and platelet counts, plasma C3a des Arg and C5b-C9 levels. The structure modification did not alter functional performances as indicated by the lack of statistically significant differences in clearance values between the two membranes. In in vitro experiments performed with normal washed and whole blood recirculated in a closed circuit demonstrated the presence of a plasma-dependent, complement-independent mechanisms responsible for the triggering of PAF synthesis and release with AN-69 but not SPAN membrane. PAF was extractable from the inner and outer side of both polyacrylonitrile membranes (AN-69: inner, 4.9 +/- 0.5 ng/ml; outer, 0.1 +/- 0.05 ng/ml; SPAN: inner, 5.5 +/- 0.6 ng/ml, outer: 3.3 +/- 0.7 ng/ml, SPAN vs. p < 0.001), suggesting that absorption may be relevant with both membranes.

S-protein/vitronectin participates in the regulation of complement-mediated lysis by incorporating into the membrane attack complex C5b-9. Subsequently, soluble SC5b-7 and SC5b-9 macromolecules are not inserted into the membrane lipid bilayer nor induce lytic pore formation. Using a dot blot binding assay, it was determined that soluble S-protein/vitronectin interacted with immobilized C5b and C8 among the five separately immobilized components (C5b, C6, C7, C8, C9) of the membrane attack complex. Those interactions imply a new model of the formation of the complement membrane attack complex and its regulation by S-protein/vitronectin.

Heymann nephritis is an extensively studied experimental model of human membranous nephropathy, a currently barely treatable glomerular immune disease. Several basic concepts were discovered by the study of this experiment disease, such as in situ formation of immune deposits, and the roles of the complement system and of oxygen radicals in the development of proteinuria. The major goal of our studies is to develop specific therapies for the experimental and eventually also for the human disease, based on the detailed knowledge of the molecular pathogenic mechanisms. The target of immune deposit forming antibodies was identified as a large membrane glycoprotein with structural similarities with the LDL-receptor. This protein serves as polyspecific receptor, and its intriguing properties in different organs have developed into a separate area of research. Sofar the precise amino acid sequences of several epitopes for the nephritogenic antibodies were identified, thus offering the unique possibility to develop precisely targeted specific therapeutic strategies. Here we link the mechanisms of immune deposit formation with the development of proteinuria.

Calcium-loaded red blood cells (RBCs) previously have been shown to have an increased sensitivity to complement-mediated hemolysis and particularly to lysis mediated by the C5b-9 membrane attack complex (MAC) of complement. Because RBCs exposed to 2-aminoethylisothiouronium bromide (AET) also have been shown to be particularly sensitive to the MAC, a direct comparison of calcium-loaded and AET-treated RBCs was performed. Calcium-loaded and AET-treated RBCs shared a marked increase in sensitivity to lysis by the MAC in two different assays. However, measurements of C5b-7 and C9 binding suggested that different mechanisms were responsible. AET-treated RBCs showed an increase in C9 binding and an increased C9/C7 ratio consistent with functional loss of CD59/membrane inhibitor of reactive lysis (MIRL). In contrast, calcium-loaded RBCs had minimally increased C9 binding that resulted in C9/C7 ratios that were less than those for untreated RBCs, suggesting that CD59/MIRL inactivation had not occurred. When RBCs were incubated in acidified serum, AET-treated cells demonstrated a marked increase in C3b binding and hemolysis that was observed in neither control nor calcium-loaded RBCs. These results suggest that the underlying lesions responsible for an increase in susceptibility to complement-mediated hemolysis are different for calcium-loaded and AET-treated RBCs.

Vitronectin (complement S-protein), a multifunctional glycoprotein, inhibits complement-mediated cytolysis at two identified stages of terminal complement complex (TCC) formation: blocking of C5b-7 membrane binding, and prevention of C9 polymerization. However, the functional domain(s) of vitronectin involved in these reactions remains incompletely defined. In order to identify the complement inhibition site, a 12-kD heparin binding fragment and two other internal fragments (53 kD and 43 kD) of vitronectin were isolated after cyanogen bromide (CNBr) treatment of the native molecule. Potent inhibition of guinea pig erythrocyte (GPE) reactive lysis was demonstrated with native vitronectin, total CNBr digest and the 53-kD and 43-kD fragments, but only very poorly by the heparin binding 12-kD peptide. Similarly, the 43-kD fragment blocked the binding of C5b-7 to immobilized vitronectin, whereas the 12-kD fragment had no effect. These data localize the C5b-7 binding site to a 43-kD internal region. Further characterization of the fragments was carried out in an assay which detected C9 polymerization in the presence of C5b-8. Polymerized material was separated by PAGE, detected by autoradiography and quantified after excision from the gels. Results showed that polymerization did not occur in the presence of the 53-kD and 43-kD fragments. However, the 12-kD heparin binding fragment had no effect. It is proposed that prevention of C5b-8-induced C9 polymerization resides at a site in an internal region of the vitronectin molecule.

Ab deposition, whether by reaction with the specific Ag or by preformed immune complexes, is followed by activation and deposition of complement components. Tissue destruction is observed in the Ab- and complement-induced lesions. The proteolytic enzyme plasmin is thought to participate in the Ab- and complement-mediated organ pathology. Plasmin is generated from plasma-derived plasminogen by cell-derived plasminogen activators (PAs). Two types of PAs are known, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated whether the PA system and the complement system can interact to promote local plasmin generation. Among the terminal complement components C5b6, C7, C8, and C9, the nonenzymatic component C7 is a plasminogen-binding protein. Radioligand binding studies revealed that the isolated component, as well as C7 after its incorporation into the terminal complement complex C5b-9, can bind plasminogen. Binding was inhibited by the lysine analogues 6-aminohexanoic acid and tranexamic acid, implicating the lysine binding sites of plasminogen into the binding interaction. tPA-mediated plasminogen activation was enhanced in the presence of C7. Based on these findings, an interaction is proposed between the complement system and the plasminogen activator system; a mechanism that may focus plasmin activity to structures that have been tagged by Ab and complement deposition.

The resistance of Aeromonas salmonicida to complement-mediated killing was investigated by using different strains and their isogenic mutants that had been previously characterized for their surface components. We found that the classical complement pathway is involved in serum killing of susceptible A. salmonicida strains, while the alternative complement pathway seems not to be involved. All of the A. salmonicida strains are able to activate complement, but the smooth strains (with or without the A-layer) are resistant to complement-mediated killing. The reasons for this resistance are that C3b may be bound far from the cell membrane and that it is rapidly degraded; therefore, the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants are serum sensitive because they bind more C3b than the smooth strains, and if C3b is not completely degraded, then the lytic complex (C5b-9) is formed.

The complement-regulatory factor C8 binding protein (C8bp) was first identified on human erythrocyte membranes by its affinity for the complement component C8 and its ability to inhibit lysis by homologous complement. Cultured human glomerular mesangial or epithelial cells (GEC) expressed C8bp on the cell surface and in the cytoplasm. Following stimulation of the glomerular cells with interleukin 1 beta, C5b-9 or with endotoxin, a transient, protein synthesis-independent increase in C8bp surface expression was seen. Blocking of C8bp function with F(ab)2 fragment of an antibody to C8bp rendered GEC susceptible to complement-mediated killing, indicating that C8bp contributes to the cellular defense against complement attack.

Complement activation properties of ricin holotoxin, its A or B chain were assessed in two enzyme immunoassays (EIA). One was specific for C3 activation products and the other detected the terminal SC5b-9 complement complex (TCC) and thus determined activation of the initial and terminal part of the complement pathways, respectively. Ricin and its A and B chains were incubated with normal human serum or EDTA-serum. It was found that ricin B chain activated both the initial and terminal complement pathways in a time- and concentration-dependent fashion, whereas the A chain and the holotoxin did not.

The terminal complement complexes C5b-7, C5b-8 and C5b-9 are able to generate nonlethal cell signals. One universal consequence of a cell being targeted by C5b-8 or C5b-9 is an influx of Ca2+. In addition, other second messengers, including cAMP, inositol phosphate intermediates and arachidonate metabolites, are generated by the terminal complement complexes in specific cell types. In vivo, terminal complement complexes have been found in a wide variety of inflammatory processes in humans and in experimental animal models. Some of these models of inflammation putatively induced by terminal complement complexes have been tested in complement-deficient animals, and indeed no inflammation results, which supports the critical role of the terminal complement complexes in the pathogenesis of the lesion.

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer.

It was earlier found that hydroxyl radicals (OH.) generated in a fluid phase system, convert human C5 to an activated, C5b-like form without cleavage. For the conversion catalytic amounts of divalent iron were necessary; upon their oxidation OH. are generated. In view of the extraordinary reactivity of OH. their specific effect on C5 was surprising. It has now turned out that C5 binds iron, about 13-15 Fe ions per molecule C5. C5 was fully bound to iron-loaded Chelating Sepharose and in this way removed form the medium. Only at the sites where iron is bound, is OH. generation possible. The half-life of OH. is extremely short and hence the radicals can act on C5 only in the immediate environment of the binding sites for iron. In this way the oxidative change of C5, induced by the radicals, is restricted to some sites on the molecule determined by the iron binding. The hydroxyl radical scavenger, dimethylsulfoxide (DMSO), is unable to interfere with the conversion of C5 by OH. generated on iron-Sepharose, it does not reach the site of reaction in that system. In fluid phase systems DMSO does inhibit the conversion of C5 to C5b-like C5.

Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore-forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.

Three cases of bilateral renal hypoplasia with oligomeganephronia are reported. In all three patients urine abnormalities were pointed out by annual urine screening for school-children and they showed mild or moderate deterioration of renal function. One patient had repeated episodes of urinary tract infection, and bilateral vesico-ureteral reflux (grade III) was found by the voiding cystourethrography. Light microscopic findings of renal biopsy specimens were similar in three cases; significant hypertrophy of the glomerulus and tubulus; mild mesangial expansion, and interstitial changes. Further histological examination which was done in one case showed widespread foot process fusion, mesangial interposition and membrane-like structures by electron microscopy, and mesangial IgA deposition by immunofluorescence. Three was no alteration in immuno-staining pattern for basement membrane components, including type VI and V collagens, laminin and fibronectin, in the glomeruli.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.

The present study using direct immunofluorescence with monoclonal antibodies to C5b-9 complex-related antigens was undertaken to determine whether complement activation in Henoch-Schönlein purpura (HSP) causes assembly of the membrane attack complex of complement (MAC) in skin and nephritis lesions. The deposition of C5, C6, C7, C8, C9, and C5b-9 neoantigens was noted in the vascular walls of papillary dermis and/or subpapillary dermal plexus of the vessels in 11 out of 15 patients with HSP. Their presence in vessel walls indicates complement activation which leads to terminal complement activation. There were small deposits of S protein at the same sites in three of the 11 skin specimens. Thus, the majority of C5b-9 demonstrated in HSP skin was the cytolytically active C5b-9 complex, MAC. Granular deposits of C5b-9 related antigens without S protein were also found in the capillary walls and mesangium of the glomeruli of two out of four specimens from patients with HSP nephritis; in the other two S protein was colocalized with the deposition of C5b-9. The results of the present study indicate that complement activation leading to generation of MAC may possibly be involved in the pathogenesis of vascular injury in a significantly large number of skin lesions and of HSP nephritis.

Measurement of the complement activation products C1s:C1-inh, C3bP and C5b-9 by ELISA in plasma samples from normals, rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE) patients showed significantly elevated levels in the two patient groups (P less than 0.0001 for C1s:C1-inh, C3bP and C5b-9) compared to normals. In seropositive RA patients there were significant correlations between the levels of the three complement activation complexes and IgM-RF, IgG-RF and IgA-RF. However, IgM-RF did not interfere with any of the ELISA systems. Mean levels of C1s:C1-inh, C3bP and C5b-9 were the same in paired plasma and synovial fluids; however, C3bP levels in the paired samples did not correlate with one another by rank. Our conclusions are that: (a) elevated plasma levels of these complement activation products are detectable in rheumatic diseases; (b) plasma levels of these complement activation products are related to Rheumatoid factor (RF) levels in seropositive RA patients; and (c) IgM-RF does not influence these solid-phase ELISA procedures.

With conventional staining techniques it was nearly impossible to differentiate with certainty between early ischemic myocardial lesions, autolytic changes and intact myocardium without a nuclear section. In 1986 it was introduced an immunohistochemical method, which presented C5b-9 Complement Complex as a necrosis marker in cryostat sections. Our further development of this process has resulted in a technique for application on routine paraffin sections: C5b-9 Complement Complex is particularly well-suited for the exact and autolytically insensitive presentation of disseminated necroses of fiber bundels and single fibers of myocardium. The application of the technique on paraffin sections will lead to the use of the same in forensic medicine routine diagnostics of early ischemic myocardial lesions, even in cases of "sudden" cardiac death.

The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.

Radioiodinated hexanoyldiiodo-N-(4-azido-2-nitrophenyl)tyramine (HNT) was employed as a photosensitive membrane-restricted probe to establish whether terminal complement proteins insert into membrane bilayers during cytolysis. The system studied consisted of natural membranes carrying intermediate (C5b-8) or fully assembled (C5b-9) cytolytic complexes of human complement prepared by two different methods. In one method, C5b-8 and C5b-9 were assembled on membranes de novo by incubating rabbit erythrocytes with C9-depleted or whole human serum, respectively. The probe was partitioned into lipid bilayers of the resulting MC5b-8 and MC5b-9 membranes by post-addition of HNT. Membranes were irradiated and photolabeled C5b-8 and C5b-9 were extracted, purified, and analyzed on polyacrylamide gels. Those constituents labeled by HNT within each complex were identified by autoradiography. The second method involved pre-addition of HNT to membranes carrying the precursive C5b-7 complex and subsequent conversion to MC5b-8 and MC5b-9 by addition of exogenous C8 and C9. After irradiation, C5b-8 and C5b-9 were again purified and analyzed for the presence of photolabel. Results from both methods were similar and indicated all constituents of each complex are labeled to a measurable extent. However, the C8 alpha subunit was predominantly labeled in C5b-8 and both C8 alpha and C9 were predominantly labeled in C5b-9. Because labeling by HNT is specific for intramembrane structural domains of proteins, these results provide direct evidence that constituents of terminal complement complexes insert into the lipid bilayer of cell membranes during lysis. Further, we conclude that in these complexes, C8 alpha and C9 are the primary contributors of inserted peptide domains.

Earlier studies have shown that sequential treatment of resealed erythrocyte ghosts with C5b6, C7, C8, and C9 leads to insertion of hydrophobic peptides from these complement proteins into the membrane and assembly of transmembrane channels. The number of molecules of each of the proteins required for assembly of the membrane-associated channel structure was evaluated by measuring the quantitative relationship between the doses of the individual proteins and the release of two trapped markers, sucrose and inulin, from ghosts after channel formation. The incubation period was sufficient to attain equilibrium of marker distribution between the ghosts and the extracellular fluid. Two markers of different size (sucrose and inulin, 0.9 and 3 nm molecular diameter, respectively) were used in order to develop information on the molecular composition of small and large channels, respectively. We found that participation of C5b6, C7, and C8 in channel formation displayed one-hit characteristics, regardless of marker size. By contrast, the participation of C9 was one-hit with respect to the sucrose marker, whereas with respect to the inulin marker the C9 reaction was multi-hit. Our results are compatible with the view that these markers are released through a channel structure in the membrane that is a monomer of C5b--9 of the composition C5b61 C71C81C9n, in which n = 1 for channels permitting passage of sucrose and n = 2 for channels allowing transit of inulin.

We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated 86Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

The authors present circumstantial evidence for the involvement of reactive hemolysis, i.e., C3-independent binding of the cytolytic C5b-9 complement complex to bystander red cells (RBC), in a case of intravascular immune hemolysis. Fresh serum obtained from a 6-year-old patient during the hemolytic episode, but not obtained thereafter, induced C5b-9-dependent hemolysis of human RBCs but the indirect C3 antiglobulin test remained negative. Particles (presumably RBC ghosts) isolated from the patient's plasma anticoagulated with EDTA at the peak of hemolysis were coated with C5b-9 complexes, whereas the direct antiglobulin test was strongly positive for IgA, only weakly positive for IgG, and negative for C3. Moreover, neither the autoantibodies isolated by elution (IgG plus IgA), nor free serum autoantibodies (IgA alone) activated complement in vitro. Additionally, serum samples collected later during the 12-month period of observation contained normal levels of C3, C4, C8, and C9, but markedly reduced levels of C7. These serums all produced strong reactive lysis in agarose plates, but not in test tubes. These results appear compatible with the working hypothesis that the intravascular hemolytic episode in this patient might have arisen through a local initiation of complement activation with subsequent C3-independent binding of C5b-9 to and hemolysis of bystander RBCs.

Age-dependent changes in the expression of Fc receptors (FcR) for different isotypes of immunoglobulins and receptors for C3b, C5b and C3bi fragments of complement on the membranes of peritoneal macrophages were studied with mice of different ages. An age-related increase in expression of Fc receptors for IgM, IgE, IgA, IgG2b and IgG3, and a decrease in the expression of Fc receptors for IgG1 was observed. The expression of FcR on macrophages of donors of different ages corresponded with Fc-receptor mediated phagocytosis. The highest number of C3b-binding macrophages was found in aged mice, in contrast to low numbers of C3bi-binding macrophages at this age. The percentage of C5b-binding macrophages was lowest in adult animals. We also observed effective inhibition of binding of the C3b component of complement by preincubation of macrophages with aggregated IgG and vice versa. These observations suggest that fluctuation in expression of Fc but not C receptors may be important to the generalized changes that occur in macrophage function during development and ageing.

By immunizing mice genetically deficient in C5 we were able to obtain a group of monoclonal antibodies to rabbit C5 that cross-react with C5 from a wide variety of mammalian sera, including mouse. The specificity of the monoclonal antibodies was against native C5 and C5b but not C5a. The antibodies strongly inhibit the expression of C5 hemolytic activity. We suggest that these monoclonal antibodies will be useful for studying C5 as well as providing a way to selectively deplete C5 from plasma in vitro or in vivo.

We examined the role of the complement system in the pathogenesis of dermatomyositis. Using an antibody against the neoantigens of the terminal C5b-9 membrane attack complex, we performed immunocytochemical studies that localized this complex to the intramuscular microvasculature (arterioles and capillaries) of muscle biopsy specimens from 10 of 12 patients (83 percent) with childhood dermatomyositis and 5 of 19 patients (26 percent) with adult dermatomyositis. Fifty-two control specimens, including 14 from patients with polymyositis and 12 from patients with denervation atrophy (a condition known to be associated with necrotic capillaries), showed no deposition of membrane attack complex in the microvasculature. These findings indicate that the complement system is deposited, bound, and activated to completion within the intramuscular microvasculature of patients with dermatomyositis. In addition to providing further evidence for the presence of vasculopathy in dermatomyositis, these findings suggest a primary role for complement in mediating vessel injury in the disease, particularly in its childhood form.

Immunoprecipitates formed in conventional electroimmunoassays were solubilized by brief immersion of the agarose gels in sodium dodecylsulfate. The proteins were then transferred to nitrocellulose sheets by electroblotting. The blotted proteins were readily amenable to analyses by non-precipitating monoclonal antibodies and the immunoblots were developed with second antibody/biotin-streptavidin-peroxidase staining. The electroimmunoassay-immunoblot (EIA-IB) method is of value in (1) specificity assays of monoclonal antibodies in crossed immunoelectrophoresis; (2) analysis of specific molecular interactions between proteins; (3) rapid screening and simple identification of monoclonal antibodies by line immunoelectrophoresis-immunoblotting.